Rough-skinned newts () use tetrodotoxin (TTX) to block voltage-gated sodium (Na) channels as a chemical defense against predation. Interestingly, newts exhibit extreme population-level variation in toxicity attributed to a coevolutionary arms race with TTX-resistant predatory snakes, but the source of TTX in newts is unknown. Here, we investigated whether symbiotic bacteria isolated from toxic newts could produce TTX. We characterized the skin-associated microbiota from a toxic and non-toxic population of newts and established pure cultures of isolated bacterial symbionts from toxic newts. We then screened bacterial culture media for TTX using LC-MS/MS and identified TTX-producing bacterial strains from four genera, including , , , and . Additionally, we sequenced the Na channel gene family in toxic newts and found that newts expressed Na channels with modified TTX binding sites, conferring extreme physiological resistance to TTX. This study highlights the complex interactions among adaptive physiology, animal-bacterial symbiosis, and ecological context.